Methods and compositions for performing analytical operations

ABSTRACT

Methods for performing analytical reactions and compositions for use in such methods, where the methods have reduced signal levels deriving from non-specific adsorption of detected reagents to other components of the analytical method, e.g., other reagents, solid phase components, vessels, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/909,797, filedJun. 4, 2013, which claims the benefit of priority to U.S. Ser. No.61/655,833, filed Jun. 5, 2012, which are herein incorporated byreference in their entireties for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

In performing analytical reactions, e.g., analyzing chemical,biochemical, biological or other reactions, a common difficulty ismaximizing the signal to noise ratio, or the ratio of relevant detectedevents from irrelevant or less relevant detected events that areindistinguishable from or otherwise interfere with the relevant detectedevents. The less relevant detected events may derive from a variety ofsources, including, e.g., ambient concentrations of reactants, built updetectable products, interfering signals from other unrelated componentsof the analysis, e.g., solution contents, reaction vessel interference,and even instrument originating background signal levels. One majorsource of noise in many analytical reactions comes from the non-specificassociation of signal producing reaction components with components ofthe reaction of interest. This includes, for example, association oflabeled reaction components, products etc., with solid phase componentsof an analytical reaction, e.g., beads, surfaces, or the like, as wellas association with other reaction components, e.g., enzymes or otherproteins, nucleic acids, cells, or the like.

In addition to reduction of background noise, enzymatic reactions sufferfrom aberrant enzyme behavior, e.g., changes in catalytic rate, pausing,dissociating, premature termination, changes in error profile, reducedrates of substrate binding and/or translocation, and the like. Forexample, in polymerase-mediated sequencing-by-synthesis reactions,polymerases have been observed to pause for extended periods of timebefore resuming normal nucleotide incorporation. In some instances,polymerase kinetics change depending on the type of nucleotide analogbeing incorporated. For example, some nucleotide analogs areincorporated at a slower rate than other nucleotide analogs, or theincorporation rate is highly variable during the course of a reaction.These behaviors have also been associated with shortened readlength anda worsening of other measures of sequencing performance, e.g., errormetrics and overall accuracy. As such, preventing or reducing aberrantenzyme behavior in enzyme-catalyzed analytical reactions would bebeneficial, e.g., to enhance enzyme performance and improve datageneration.

Accordingly, it is desirable to reduce the overall level of backgroundnoise in analytical reactions, and particularly to reduce suchbackground that derives from non-specific association of detectablereaction components with other components of the analysis. It is alsodesirable to increase or enhance the fluorescence intensity and/orimprove fluorophore photostability. Yet further, it is desirable toprevent or reduce aberrant enzyme behavior, e.g., in order to increasereadlength and accuracy of analytical reactions such aspolymerase-mediated single molecule sequencing. The present inventionprovides solutions to these and other problems.

BRIEF SUMMARY OF THE INVENTION

The present invention is generally directed to methods and compositionsfor performing single molecule analytical reactions in the presence ofadditives.

In one aspect, the present invention provides a method of conducting ananalytical reaction, where that method includes the steps of providing areaction mixture comprising a first reaction component coupled to asurface of a solid support and a second reaction component having adetectable property and conducting the analytical reaction in thepresence of at least one additive.

In a further embodiment and in accordance with the above, the presentinvention includes compositions for use in methods of conductinganalytical reactions comprising at least one additive selected from:cholesterol, cholic acid, an amino acid, an organic solvent, and acompound comprising an aromatic ring and two —NR₂ substituents.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising taurocholic acid asan additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising a sulfonatedtaurocholic acid derivative as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising arginine as anadditive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising dimethylformamide(DMF) as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising dimethylacetamide(DMA) as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprisingN-methyl-2-pyrrolidone (NMP) as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprisingN,N-dimethyl-m-phenylenediamine (DMMP) as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprisingN,N-dimethyl-m-phenylenediamine (DMPP) as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising N-formylmorpholine(FMP) as an additive.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes compositions comprising an additive that is asulfated cyclodextrin

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes methods in which the first reactioncomponents is provided disposed on the solid support as a singlemolecule or single molecular complex that is resolvable from other firstreaction component molecules or molecular complexes on the solidsupport.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes methods in which the solid support comprisesa silica based substrate.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes methods in which the solid support comprisesa charged surface, and the additive comprises charged groups that areoppositely charged to the charged surface.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes methods in which the solid support comprisesa hydrophobic surface and the additive comprises a hydrophobic group.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes methods in which the solid support comprisesa hydrophilic surface, and said additive comprises hydrophilic groups.

In an exemplary embodiment and in accordance with any of the above, thepresent invention includes methods in which the second reactioncomponent comprises a fluorescent labeling moiety.

In a further embodiment, the present invention includes compositionscomprising as additives substituted cyclodextrin compositions, whichcompositions improve the overall functioning of those analyticalsystems. In particular, the methods of the invention relate to singlemolecule analytical reactions such as sequencing and related reactionsusing such substituted cyclodextrin compositions. Also provided arenovel substituted cyclodextrin compositions for use in such methods andin other applications.

In an exemplary embodiment, the methods of the invention includeanalyzing a reaction. These methods comprise providing a reactionmixture comprising a first reaction component coupled to a surface of asolid support and a second reaction component having a detectableproperty in the presence of a sulfated cyclodextrin having thestructure:

where n is from 6 to 12; R₁ is a group that is non-attractive to thesecond reaction component; R₂ and R₃ are associative groups to thesurface of the solid support; and the sulfated cyclodextrin is presentat an isomeric purity of at least 80%. An interaction is detectedbetween the first reaction component and the second reaction componentby detecting the detectable property of the second reaction componentassociated with the first reaction component.

In one embodiments, the invention provides novel compositions, such ascyclodextrin compositions, comprising the formula:

where n is from 6 to 12; R₁ comprises a negatively charged group; R₂ andR₃ are selected from CH2-acyl, diethylacetamide, dipropylacetamide,morpholino, piperazine, piperidine, pyrrolidine, and oxazolidine; andthe sulfated cyclodextrin is present at an isomeric purity of at least80%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary cyclodextrin compounds according to the presentinvention

FIG. 2 shows a synthetic scheme for a substituted cyclodextrin compound,heptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin (alsointerchangeably referred to as SBCD-MOR, Cyclo-Mor, andcyclodextrin-morpholino).

FIG. 3 shows a synthetic scheme for an alternative substitutedcyclodextrin compound.

FIG. 4 shows a comparison of raw sequence read accuracy in the absenceand presence of substituted cyclodextrin compounds (“Cyclo-Mor” refersto cyclodextrin-morpholino orheptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin).

FIG. 5 shows a comparison of average readlengths for determinedsequences in the absence and presence of substituted cyclodextrincompounds (“Cyclo-Mor” refers to cyclodextrin-morpholino orheptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin).

FIG. 6 illustrates exemplary compounds of use in compositions of thepresent invention.

FIG. 7 illustrates examples of polymerase pausing in sequencing data.

FIG. 8 illustrates an example of polymerase pausing depicted by anaccounting stall report metric.

FIG. 9 shows data on the effect of arginine on polymerase pausingdepicted by an accounting stall report metric. The X and Y axis of eachof the graphs in FIG. 9 are the same as those in FIG. 8.

FIG. 10 shows data on the effect of DMMP and DMPP on polymerase pausingdepicted by an accounting stall report metric. The X and Y axis of eachof the graphs in FIG. 10 are the same as those in FIG. 8.

FIG. 11 shows data on the effect of arginine and DMMP on pausingreduction an polymerase rate increase for different polymerases.

FIG. 12 shows data on the effect of FMP on polymerase pausing. The X andY axis of each of the graphs in FIG. 12 are the same as those in FIG. 8.

FIG. 13 shows data on non-specific interaction or “sticks” betweendye-labeled nucleotides and a surface in solutions of differing ionicstrength.

FIG. 14 shows data on the effect of increasing DMF concentration onnon-specific interactions between nucleotide analogs and a surface.

FIG. 15 shows data on the effect of organic solvent during sequencing.

FIG. 16 illustrates exemplary compounds of use in compositions of thepresent invention.

FIG. 17 shows data on the effect of cyclodextrin compounds of theinvention on polymerase rate.

FIG. 18 shows data on the effect of different concentrations of cholicacid on polymerase rate.

FIG. 19 shows data on the effect of different concentrations of cholicacid on polymerase rate.

FIG. 20 shows data on the effect of cholic acid on read length.

FIG. 21 shows an exemplary compound for use in compositions in thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, phage display, anddetection of hybridization using a label. Specific illustrations ofsuitable techniques can be had by reference to the example herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals such as Genome Analysis: A Laboratory ManualSeries (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: ALaboratory Manual, PCR Primer: A Laboratory Manual, and MolecularCloning: A Laboratory Manual (all from Cold Spring Harbor LaboratoryPress), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York,Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, compositions, formulations andmethodologies which are described in the publication and which might beused in connection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

Overview

The present invention includes both methods for performing analyticalreactions and compositions for use in such methods, where the methodshave reduced background signal levels deriving from non-specificadsorption of detected reagents to other components of the analyticalmethod, e.g., other reagents, solid phase components, vessels, etc. Themethods may in addition or in the alternative also result in a reductionin aberrant enzyme behavior, e.g., lower or variable enzyme activity(e.g., reduced catalytic rates, less efficient substrate binding, slowerincorporation and/or translocation rates, or altered error profiles(e.g., more or different types of errors)), polymerase pausing (i.e.,extended halting of incorporation of nucleotides into a nascentnucleotide strand by a polymerase), or other changes in the kinetics ofcatalytic activity. For example, interpulse duration (IPD) is onemeasure of enzyme activity that can be positively affected by additionof one or more of the additives described herein. Generally speaking andin certain embodiments, IPD is the period of time followingincorporation of a nucleotide (e.g., and exit of a label from thereaction site) and prior to binding of a subsequently incorporatednucleotide in a sequencing-by-synthesis reaction.

In certain embodiments, the compositions of the present inventioninclude additives that are able to reduce non-specific adsorption ofreagents to other components of the analytical methods, includingwithout limitation cyclodextrin, FMP, and derivatives thereof. Suchcompositions are further described herein and some of which are alsoprovided in U.S. Pat. No. 8,124,359; Zhou, et al. (2008) Proc. of SPIE7267:726719(1-10); and Garcia, et al. (2005) Electrophoresis26(3):703-709, all of which are incorporated herein by reference intheir entireties for all purposes.

In further embodiments, additives of use in methods and compositions ofthe invention include one or more moieties, including lipophilicmoieties, that are able to provide an anchor to a surface (includingwithout limitation a glass surface). Such additives in furtherembodiments also include a water solubilizing group (including withoutlimitation a sulfonyl).

In certain exemplary embodiments and in accordance with any of theabove, additives of the invention include cholesterol and compounds withsimilar structures to cholesterol, such as cholic acid and derivativesthereof.

In further exemplary embodiments and in accordance with any of theabove, additives of the invention include organic solvents, surfactants,and/or polymer additives, including without limitation dimethylformamide(“DMF”), polyethylene glycol, and derivatives thereof. Other suchadditives are provided in U.S. Pat. No. 6,242,235, which is incorporatedherein by reference in its entirety for all purposes.

In further aspects of the invention, additives include molecules thatreduce aberrant polymerase activity, including but not limited to low orvariable catalytic rates (e.g., rates of incorporation and/ortranslocation), changes in error profile, reduced substrate binding,and/or “pausing” during single molecule real-time sequencing, includingSMRT® sequencing (described in, e.g., U.S. Pat. Nos. 6,399,335,6,056,661, 7,052,847, 7,033,764, 7,056,676, 7,361,466, 7,416,844, thefull disclosures of which are incorporated herein by reference in theirentirety for all purposes and in particular for all teachings related tosingle molecule sequencing methods). Polymerase pausing can limitreadlength in sequencing methods, because the polymerase will go throughperiods of quiescence/non-functioning during which normal nucleotideincorporation is paused. Typically, such polymerase pausing in apolymerization reaction represents a cessation of polymerase activitythat is at least 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or100-fold longer than the average IPD. Other types of aberrant polymeraseactivity include changes to catalytic or enzymatic rates (e.g.,substrate binding, substrate incorporation, and/or translocation rates),error profile, processivity, and the like. As will be appreciated, anyof the additives described herein as reducing non-specific absorptionand/or binding of reactants may also serve to reduce aberrant polymeraseactivity, and vice versa.

In certain exemplary embodiments and in accordance with any of theabove, additives that reduce polymerase pausing include withoutlimitation amino acids (including charged amino acids such as arginineand glutamate), additives comprising an aromatic ring and two —NR2substituents, and organic solvents. In other embodiments, such additivesinclude DMMP, DMPP, and FMP or other morpholine derivatives.

In other exemplary embodiments and in accordance with any of the above,additives that mitigate aberrant polymerase activity include withoutlimitation cholesterol and compounds with similar structures tocholesterol (e.g., cholic acid, taurocholic acid, etc.), poly dToligonucleotides, and cyclodextrin derivatives (e.g., heptasulfonatedcyclodextrin). For example, in reactions that includepolymerase-mediated nascent strand synthesis using nucleotide analogscomprising bulky groups (e.g., a detectable label such as one or morefluorescent dye molecules attached to the nucleotide), the polymerasehas a tendency to slow down such that the time between incorporationevents (“interpulse duration” or IPD) is lengthened relative to the samereaction using less bulky nucleotide analogs. The increase in IPD canresult in a decrease in overall read length. Addition of one or more ofthese additives reduces the IPD and increases the overall read length inthe presence of such bulky nucleotide analogs. In certain preferredembodiments, such bulky nucleotide analogs comprise FRET labels, e.g.,linked to a phosphate group that is removed during incorporation of thenucleotide monophosphate into a nascent strand by the polymerase. Inother embodiments, addition of these additives not only decreasesaverage IPD, but also reduces IPD variability, e.g., between differentreactions. In yet further embodiments, possibly due to the effects onIPD, reaction rates are increase in the presence of these additives. Forexample, cholic acid and derivatives thereof increase overall polymeraserate in polymerization reactions. Other such additives are also providedin U.S. Patent Publication No. 2010/0035767 and Golovanov, et al. (2004)JACS 126:8933-8939, which are incorporated herein by reference in theirentireties for all purposes.

In accordance with any of the above, methods of the invention utilizecompositions comprising one or more additives described herein inconcentrations of about 0.01-200, 0.1-190. 1.0-180, 5-170, 10-160,15-150, 20-140, 25-130, 30-120, 35-110, 40-100, 45-90, 50-80, 55-70, and60-65 mM. In other embodiments, methods of the invention utilizecompositions comprising one or more additives described herein inconcentrations of about 0.01-200, 0.1-190. 1.0-180, 5-170, 10-160,15-150, 20-140, 25-130, 30-120, 35-110, 40-100, 45-90, 50-80, 55-70, and60-65 μM.

Although the following sections disclose exemplary additives of use inthe present invention separately, the current invention also encompassesany combination of the additives disclosed herein as well as those knownin the art.

Cholesterol and Similar Compounds

As discussed above, one characteristic of some embodiments of additivesof use in the present invention are that these additives include amoiety that is able to provide an anchor to surfaces. These additivesmay in further embodiments also carry a water solubilizing group.Exemplary embodiments of additives with these characteristics includecholesterol and compounds with similar skeletons to cholesterol, such ascholic acid. Such additives can include a liphophilic moiety that servesas an anchor to surfaces (including glass surfaces or exposed areas ofcoated glass surfaces). Such additives can also include a watersolubilizing group.

In a specific embodiment, cholic acid with a water soluble subunit isused as an additive in methods and compositions of the invention. In afurther embodiment, sulfonic cholic acid derivatives are used. In astill further embodiment, sulfonated cholic acid derivatives of use inthe present invention include without limitation the compoundsillustrated in FIGS. 6 and 21. As is also depicted in FIG. 6, thesulfopropyl moiety can be attached to any of the hydroxy moieties in thecholic acid derivatives of use in the present invention. In yet furtherembodiments, taurocholic acid is used as an additive in compositions ofthe present invention. In a further embodiment, the taurocholic acidused is in a potassium salt form.

In further embodiments, methods and compositions of the inventionfurther have the effect of reducing the interpulse distance oflipophilic FRET analogs. Data showing the effects of compositions usingcholic acid on interpulse duration are shown in FIGS. 18 and 19. Effectof cholic acid on readlength, which is reflective of IPD, is shown inFIG. 20.

In accordance with any of the above, methods of the invention utilizecompositions comprising one or more cholesterol or cholic acidderivatives described herein in concentrations of about 100-500,110-400, and 120-300 μM.

Organic Solvents

One potential cause for non-specific binding of dye-labeled nucleotides(also referred to herein as analogs) may be through hydrophobicinteractions between the analogs and components of the reaction,including the surface upon which the reaction takes place. For example,FIG. 13 shows that non-specific interaction (“sticks”) between analogsand PEG-coated silicon dioxide surface increases with ionic strength ofthe solution.

One aspect of the present invention includes additives that can weakenthe hydrophobic interaction between analog and surface, thus reducingthe “stick” rate and errors that can result from such non-specificbinding. Organic solvents, including without limitation DMF, are of usein accordance with this aspect of the invention.

FIG. 14 shows that stick rate decreases with increasing DMFconcentration. FIG. 15 shows further studies with DMF—sequencing withDMF resulted in an accuracy gain, which can be attributed to reductionof stick pulses. The data are from an experiment in which eight chipswere sequenced at control conditions without organic solvent and anothereight chips were sequenced with 6% DMF. The figure is a cumulativedistribution function (CDF) of Z-score, in which higher Z-score meansbetter sequencing alignment or accuracy. The “p1” and “p2” in the legendshow the average data from two consecutive movies taken from each chip.Among the four CDF curves, the two from chips sequenced with DMF showhigher CDF fraction number at high-Z-score range, which means they havehigher Z score and accuracy.

In addition to mitigation of non-specific binding of dye-labelednucleotides, organic solvents can also improve the photophysicalcharacteristics and behavior of fluorescent dyes. For example, incertain aspects, the presence of one or more organic solvents improvesfluorescent dye intensity. In further aspects, addition of one or moreorganic solvents results in improved fluorophore photostability. Withoutbeing bound by theory, it is conceived that the enhancement ofphotostability itself may underlie the observed increase in dyeintensity.

Organic solvents that may be included in compositions of the inventioninclude, without limitation, tetrahydrofuran (THF), ethyl acetate,acetonitrile, dimethyl sulfoxide (DMSO), dioxane, dimethylformamide(DMF), dimethylacetamide (DMA), formamide,N,N-(2-hydroxyethyl)formamide, N-pyridine-3-formamide, formylmorpholine, methylpyrrolidone, N-methyl-2-pyrrolidone (NMP), t-butanol,ethanol, ethylene glycol, glycerol, n-propanol, isoproponal, aceticacid, and others described in the art, e.g., in U.S. Pat. No. 6,242,235,incorporated herein by reference in its entirety for all purposes.Further, detergents may be included in compositions of the invention,and some such detergents are further described in the art, e.g., in U.S.Patent Publication Nos. 2011/0312035 and 2010/0099150, both of which areincorporated herein by reference in their entireties for all purposes.Yet further, surfactants and/or polymer additives, such as polyethyleneglycol, may also be included in compositions of the invention.

Additives that Reduce Aberrant Enzyme Activity

Aberrant enzyme activity can be caused by various different conditionswithin an analytical reaction. For example, instability of an enzymecomplex, steric hindrance, charge interactions, and sticking of reactioncomponents to surfaces of a device are only a few conditions that canlead to aberrant enzyme activity, which can be detected as, e.g., loweror variable enzyme activity (e.g., reduced catalytic rates, lessefficient substrate binding, slower incorporation and/or translocationrates, or altered error profiles (e.g., more or different types oferrors)), polymerase pausing (i.e., reduced halting of incorporation ofnucleotides into a nascent nucleotide strand by a polymerase), or otherchanges in the kinetics of catalytic activity. As noted above,interpulse duration (IPD) is one measure of enzyme activity that can bepositively affected by addition of one or more of the additivesdescribed herein. In certain embodiments, IPD is the period of timefollowing incorporation of a nucleotide and prior to binding of asubsequently incorporated nucleotide in a sequencing by synthesisreaction. Polymerase pausing can also be mitigated by addition of one ormore of the additives described herein.

In certain aspects, the invention addresses increased IPD due to use ofbulky nucleotide analogs in a polymerization reaction. Long or variableIPD can limit the readlength and affect the error profile of apolymerase enzyme. For example, a polymerase that is slow to translocateand/or bind a new nucleotide analog after incorporation will notincorporate as many nucleotides during a given time period as apolymerase that has a more rapid translocation and substrate bindingrate.

In certain aspects, the invention addresses polymerase pausing, whichcan limit the readlength in single molecule sequencing methods. Forexample, many sequencing traces demonstrate periods of time in which thepolymerase enzyme ceases functioning for a period of time prior toresuming normal nucleotide incorporation. An example of polymerasepausing within a SMRT® sequencing trace is shown in FIG. 7.

Certain additives of the invention include additives that reducepolymerase pausing, thereby functioning as “pausing reducers.” Someadditives of the invention include additives that mitigate aberrantenzyme activity other than pausing, e.g., increased IPD, therebyfunctioning as “kinetic improvers.” Although the following descriptiondescribes additives of particular utility in reduction of aberrantenzyme activity, it will be appreciated that the following additives mayalso be used for reduction of background signal noise by reduction ofnon-specific adsorption of reagents to components of a reaction, as areother additives described herein. In addition, additives describedherein as being useful for reduction of background signal noise may alsobe used to reduce aberrant enzyme activity.

In accordance with any of the above, additives of the invention includeamino acids. In specific embodiments, additives of the invention includecharged amino acids such as arginine and glutamate. In exemplaryembodiments, amino acids such as arginine are included in compositionsof the invention as a bulk additive to reduce polymerase pausing.Although the following discussion focuses on arginine as an additive, itwill be appreciated that any amino acid, particularly other chargedamino acids and amino acids with similar side chains to arginine, can beused in accordance with the discussion herein.

In specific embodiments of the invention, use of arginine as an additivein compositions of the invention serves to reduce polymerase pausing.When the amount of arginine additive was increased, reduction ofenzymatic pausing was observed (FIG. 9). Without being bound by theory,one possible mechanism for action of the effect of arginine is that itscharged side-chain containing a conjugated —NR₃ moiety may beresponsible for pausing reduction. In some embodiments, arginine isincluded in compositions of the invention in concentration ranges offrom about 1 mM to 100 mM. In certain embodiments, arginine is includedin concentrations of from about 20-40 Mm. In further embodiments,arginine is included in a concentration of about 30 mM. In still furtherembodiments, arginine is included in concentrations in ranges of about1-150, 10-140, 20-130, 30-120, 40-110, 50-100, 60-90, 70-80 mM.

In further embodiments, oligopeptides of from about 2-20 amino acids areused as additives in compositions of the invention. In exemplaryembodiments, such oligopeptides include at least one arginine residue.In still further embodiments, such oligopeptides include at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 arginineresidues.

In still further embodiments, compounds similar to the side-chain ofarginine, such as guanidine and its derivatives, can also be used inaccordance with the present invention as additives. In yet furtherembodiments, compounds similar to guanidine (i.e. urea and itsderivatives) may also be used as additives.

In one aspect of this invention, compounds comprising an aromatic ringand two —NR2 substituents may be useful as additives for reducingpolymerase pausing. For example, N,N-dimethyl-m-phenylenediamine (DMMP)or N,N dimethyl-p-phenylenediamine (DMPP) both demonstrated pausingreduction (FIG. 10). In certain embodiments, such additives are presentin a concentration range of from about 0.1 mM to about 50 mM. In furtherembodiments, additives comprising an aromatic ring and two —NR₂substituents are present in a concentration range of from about 1 mM toabout 10 mM. In a yet further embodiment, such additives are present ina concentration of about 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5 mM. Instill further embodiments, additives comprising an aromatic ring and two—NR₂ substituents are present in a concentration range of 0.01-100,0.1-90, 0.5-80, 1-70. 10-60, 20-50, 30-40 mM.

In addition to pausing reduction capabilities, additives of theinvention, including additives comprising an aromatic ring and two —NR2substituents, can further serve as quenchers of the reactive tripletstate.

The effect of pausing reduction on polymerase rate by both Arg and DMMPadditives is demonstrated in FIG. 11.

In yet another aspect of this invention, morpholine derivates, includingwithout limitation N-formylmorpholine (FMP), are used as additives toreduce polymerase pausing. Decrease of pausing with increase of FMPconcentration is shown in FIG. 12. In certain embodiments, aconcentration range of from about 0.1% to 20% by volume of morpholinederivatives are used in accordance with the present invention. Infurther embodiments, 2-10% by volume concentration range is used. In ayet further embodiment, a concentration of about 2, 3, 4, 5, 6, 7, 8, 9,10% by volume is used. In a still further embodiment, a concentrationrange of from about 0.1-30, 0.5-25, 1.0-20, 1.5-15, 2.0-10, 2.5-5% byvolume is used.

In further aspects of the invention, other organic solvent additives areused as additives in compositions of the invention to reduce polymerasepausing.

In accordance with any of the above, additives of the invention includecholesterol or cholic acids and derivatives thereof. In specificembodiments, such additives of the invention include taurocholic acidand sulfonic cholic acid derivatives, cyclodextrin and derivativesthereof, and dT oligonucleotides. In exemplary embodiments, suchcompounds are included in compositions of the invention as a bulkadditive to reduce aberrant enzyme activity, and in specificembodiments, to reduce IPD, e.g., where bulky nucleotide additives areused. When measuring over a plurality of single molecule reactions, IPDvalues are a distribution, so some variability exists from each reactionto the next. However, when using certain nucleotide analogs, e.g., bulkyanalogs having large labels, e.g., FRET labels, that distribution shiftstoward longer IPD values. Inclusion of these additives within thereaction mixture pushes the distribution back toward shorter IPD values.For example, addition of cholic acid derivatives such as taurocholicacid and sulfonated cholic acid derivatives reduces IPD values forpolymerase-mediated single-molecule sequencing reactions usingnucleotide analogs comprising multi-dye constructs, e.g., FRET dyes.Similar effects are detected when poly dT oligonucleotides,cyclodextrin, or cyclodextrin derivatives are added.

Cyclodextrin

The present invention provides methods of employing cyclodextrincompositions in the performance of solid phase enzyme reactions andparticularly those solid phase reactions that utilize labeled reactants,such as fluorescently labeled reactants. For example, cyclodextrincompositions can be employed as kinetic improvers, as described above.The cyclodextrin compositions utilized in the methods of the inventionpreferably include substituted cyclodextrins that are preferably presentas substantially isomerically pure compositions.

In certain particular aspects, the methods of the invention compriseproviding an observation region for monitoring a reaction of interest,providing in the reaction region a reaction mixture that includes firstand second reactants that react in the reaction of interest, where atleast one the first and second reactants comprises a detectableproperty, and a cyclodextrin composition of the above structure, wherebythe cyclodextrin is present at a concentration that reduces non-specificinteractions between the reactant having the detectable group and otherreaction components present in the observation region.

Exemplary reactions include binding reactions, catalytic reactions, andsynthetic reactions where one component of such reaction bears adetectable property such as a labeling group or the like. Bindingreactions, for example, typically employ a first component that isimmobilized upon a solid support such as a matrix, or solid surface of asubstrate. A detectable reagent that is being tested for its ability tobind the first reagent is then exposed to the solid support, and theresulting system is washed to remove any unbound detectable reagent. Anyremaining detectable reagent is thought to be bound to the firstimmobilized component. Such binding reactions are widespread inanalyses, including for example, antibody/antigen assays, nucleic acidhybridization assays, ligand/receptor assays, and myriad others. As willbe appreciated any nonspecific adsorption of the detectable component tothe overall system, e.g., either the underlying solid support or thereaction component that is bound to the underlying substrate, will yielda level of background signal or noise that is not relevant to adetermination of specific binding. Such background noise reduces theoverall sensitivity of the analysis.

Examples of cyclodextrin compositions for use in the methods of theinvention include those compositions that have been previously describedfor use as chiral resolving agents (See, e.g., U.S. Pat. No. 6,391,862,which is incorporated herein by reference in its entirety for allpurposes and in particular for all teachings related to chiral resolvingagents). Variations on such compositions are also included within themethods and compositions of the invention, as described in greaterdetail herein.

Without being bound to a particular theory of operation, it is believedthat these compositions serve to block potential non-specific bindingsites on other reaction components, e.g., underlying substrates or solidsupports, proteins, or the like, which are believed to provideassociation sites for the detectable reagents used in these analyticaloperations, or the by-products of these reagents, contributing tointerfering signal noise. It is believed that the noise contributed bythis nonspecific interaction is reduced or eliminated by blocking thesenon-specific association sites using the compositions described herein,as shown by the consequent improvement in signal to noise ratios andother reaction quality metrics.

As noted above, exemplary structures of the compositions used in themethods of the invention include those set forth in U.S. Pat. No.6,391,862, previously incorporated herein, such as sulfatedcyclodextrins, and variations of such compositions. In general, thecompositions used in the methods described herein have the structure:

where n is from 6 to 12, R1 is a group that is non-attractive to thedetectable component of the reaction of interest, i.e., neutral orrepulsive with respect to the detectable component of the reaction ofinterest. For example, where the detectable component of the reaction ofinterest is charged, then R1 would preferably be uncharged or would beara common charge, e.g., positive to positive. Likewise, where thedetectable reaction component is hydrophobic, then R1 may behydrophilic, so as to repel the detectable reaction component, or viceversa.

For certain compositions used in the methods of the invention, R2 and R3are selected to provide an associative group with respect to thenon-specific binding sites on other reaction components that are soughtto be blocked or masked, e.g., solid support surfaces, proteins, etc.For certain applications, where the nonspecific binding sites comprisehydrophobic groups, e.g., on the surface of a substrate or other solidsupport that has hydrophobic characteristics, then R2 and R3 wouldpreferably be hydrophobic groups to associate with and block suchsurface groups from associating with detectable reaction components.Likewise, if the non-specific binding sites are charged, then R1 and R2would preferably be oppositely charged to associate with and block suchgroups.

A wide variety of charged groups may be selected to provide therequisite association or repulsion for the various groups on thecompositions described herein. For example, where any or all of R1, R2and R3 are negatively charged groups, they are preferably selected fromsuch negatively charged groups as sulfate, phosphate, carboxylate, andthe like.

Where any or all of R1, R2 and R3 are positively charged groups, theyare preferably selected from such groups as ammonium, quaternary amine,and the like.

Where any or all of R1, R2 and R3 are hydrophobic groups, they aregenerally selected from groups such as alkyl, e.g., C1-C12,hydroxyalkyl, e.g., C2-C8, a acyl, e.g., C2-C12, aryl, carbamate, athiocarbamate, carbonate, silyl, ester, or combinations thereof.

As will be appreciated, the selection of R1, R2 and R3 groups can dependupon both the nature of the nonspecific binding groups and the nature ofthe detectable reaction components, which also can be related. Inparticular, where a detectable reagent is positively charged, it cannon-specifically associate with negatively charged groups on the surfaceof a solid support. In conjunction with the invention then, the R2 andR3 groups may be selected to be positively charged groups to associatewith the negatively charged surface groups. In addition, the R1 groupwould be selected to be non-attractive to the detectable reactioncomponent. In the case of a positively charged detectable reactioncomponent, a positively charged R1 group may be used.

In order to ensure that the cyclodextrin compositions of the inventionare consistently oriented to present the desired characteristics to thedesired environment, e.g., presenting hydrophobic groups to a surfacewhile presenting a negatively charged group to the reaction mixture, itis preferable that such mixtures exist as a substantially isomericallypure composition. Preferably, the cyclodextrin composition has anisomeric purity of at least 80 mole %, with an isomeric purity of atleast 90 mole % being more preferable, and an isomeric purity of atleast 95 mole % being even more preferable. Methods for derivingcyclodextrin compounds of sufficient purity are known in the art and aredescribed, for example, in U.S. Pat. No. 6,391,862, previouslyincorporated here by reference.

In certain particularly preferred aspects, R1 is SO₃ ⁻ while R2 and R3are hydrophobic groups, such as alkyl, hydroxyalkyl, acyl, CH2-acyl, andacetamide groups, including, for example diethylacetamide groups,dipropylacetamide, and the like, as well as aryl or heteroaryl groupssuch as morpholino, piperazine, piperidine, pyrrolidine, oxazolidine,and the like.

For example, in certain particularly preferred aspects, the methods ofthe invention comprise nucleic acid analyses where the detectablereaction component comprises a nucleic acid, or nucleosidepolyphosphate, and the reaction is carried out at or proximal to asubstrate surface. Accordingly, the compositions used in such preferredmethods would typically include a negatively charged group in the R1position, such as a sulfate group. In addition, the R2 and R3 groupswould preferably be selected to associate with the substrate surface.For underivatized silica substrates that bear a substantially negativesurface charge, positively charged R2 and R3 groups would be preferablyselected. For derivatized surfaces that bear hydrophobic groups however,R2 and R3 would generally be selected from hydrophobic substituents.

Examples of compounds according to the particularly preferred aspects ofthe present invention include, e.g.,heptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin (Compound 1),and heptakis(2,3-diacetyldiethylacetamide-6-sulfato)-β-cyclodextrin(Compound 2). These compounds are illustrated with reference to FIG. 1which shows a sulfated cyclodextrin molecule with substitutions ofmorpholine and diethylacetamide for the 2′-OH and 3′-OH positions.

As discussed above, interpulse duration (IPD) is one measure of enzymeactivity that can be positively affected by addition of one or more ofthe additives described herein. In certain embodiments, IPD is theperiod of time following incorporation of a nucleotide and exit of alabeled polyphosphate, and prior to binding of a subsequentlyincorporated nucleotide in a sequencing-by-synthesis reaction. Incertain embodiments, cyclodextrin compounds of the invention affectinterpulse duration, which can be reflected in polymerase rate, as shownin FIG. 17 for various cyclodextrin compounds of the invention (whichare illustrated in FIG. 16). The following table further provides dataon the effect of various cyclodextrin compounds of the invention onreadlength, which is reflective of reduced IPD and increased polymeraserate:

SY360- Poly dT SY360-154 SY360-155 156 Median Z-score 17.92 17.34 18.2313.13 Median Accuracy 86.84% 86.91% 87.34% 81.36% Median Readlength 564bp 356 bp 504 bp 376 bp # of reads with 1274 957 974 844 Z > 3 MedianAccuracy 87.22% 87.22% 87.94% 82.57% Median Readlength 566 bp 356 bp 508bp 379 bp # of bases 662228 303565 470277 259236 with QV >=6

Additional Components and Formulations for Compositions of the Invention

As will be appreciated, in addition to the additives discussed above,compositions of the invention may further include components useful toanalytical reactions, particularly sequencing reactions. Such componentsare known in the art and can include without limitation (singly or inany combination): nucleotides or nucleotide analogs, buffers such asphosphate, citrate, other organic acids, MOPS, bis-tris propane (BTP);antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EGTA or EDTA; reducing agents such asDTT, sugars such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions such as potassium, sodium, magnesium, andstrontium; metal complexes (e.g. Zn-protein complexes); non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG),and/or coenzymes such as ATP. In certain preferred embodiments at least1, 2, 3, 4, 5, or 6 or more of the above components are used incombination with the additives described elsewhere herein.

In further embodiments, compositions of the invention include theadditives discussed herein and any combination of one or more otherreaction components known in the art and discussed for example inWO/2010/144150; WO/2009/114182; WO/2009/102470; WO/2011/112260; U.S.Pat. No. 7,767,394; U.S. Pat. No. 7,405,281; US 2012/0009567; US2007/0128133; US 2010/0167299, each of which is hereby incorporated byreference in its entirety for all purposes and in particular for allteachings related to components of compositions used in analyticalreactions such as sequencing reactions.

In still further embodiments, compositions of the invention comprisecomponents according to the following general formulation:

General Formulation

Tris-type buffer (pH 7.5-8.0) to stabilize reaction pH 20-30 mM Salt,e.g., KOAc, LiOAc, NH₄OAc, to set ionic strength, mitigate protein 120mM aggregation, and/or modify rate of catalysis by competition withcofactor (In further embodiments, multiple salts are included. In stillfurther embodiments, LiOAc is included at a lower concentration, e.g.,less than 1 mM) Enzyme cofactor; sets rate of catalysis, e.g., MgOAc orMnOAc 25-30 mM Reducing agent/photodamage mitigator, e.g., DTT, MEA (canbe used 10-40 mM separately or in combination, e.g. with DTT at 40 mMand MEA at 10 mM) Anti-pause agent (increases rate of polymerization),e.g., DMAPA, DMPP, 2.5 mM DMMP (In alternative embodiments, Arginine isused @ 20-50 mM) Anti-sticking agent 1; shortens IPDs; increases rate ofpolymerization, e.g., 120 μM taurocholic acid, cyclodextrin-morpholino,poly-dT Anti-sticking agent 2, e.g., FMP, DMF, DMA, DMSO 2-4% Chelatingagent, e.g., EDTA, EGTA (optional) 3 mM

As will be appreciated, in further embodiments, the formulation mayinclude a subset of the above listed components in any combination.

The above general formulation may further include any of the otheradditives discussed herein. In further embodiments, compositions of theinvention include one or more components of the above generalformulation in combination with other additives, including withoutlimitation additives for photodamage mitigation, such as oxygenscrubbers and triplet state quenchers, and any of the additivesdescribed for example in U.S. Pat. No. 7,993,895; U.S. Pat. No.8,252,911; U.S. Patent Publication No. 20090325260; U.S. Pat. No.8,388,982; U.S. Patent Publication No. 20120052488; U.S. PatentPublication No. 20120009567 (01-012901); and U.S. ProvisionalApplication No. 61/707,621, filed Sep. 28, 2012, each of which is herebyincorporated by reference for all purposes and in particular for alldisclosure, figures or examples related to additives to include inreaction compositions. It will be further understood that where theformulation is to be used for an analytical reaction, components of theanalytical reaction will also be present, for example, proteins (e.g.,enzymes), cofactors, substrates, competitive agents, inhibitors, agentsto initiate and/or terminate the reaction, and other components, whetherpresent at the initiation of the reaction or added subsequent to theinitiation.

In still further embodiments, compositions of the invention comprisecomponents according to the following exemplary formulations:

Exemplary Formulation 1

Bis-Tris-Propane 25 mM Stabilizes reaction pH buffer (pH 8.0) MgOAc 25mM Enzyme cofactor; sets rate of catalysis LiOAc 0.5 mM  Modifies rateof catalysis by competition with MgOAc KOAc 120 mM  Sets ionic strength;prevents protein aggregation DTT 40 mM Reducing agent; mitigates photo-induced damage DMAPA 2.5 mM  Anti-pause agent (increases polymeraserate) Cyclodextrin- 120 μM   Anti-sticking agent; increases accuracymorpholino & read length FMP 4% Anti-sticking agent

Exemplary Formulation 2

Bis-Tris-Propane 20 mM Stabilizes reaction pH buffer (pH 8.0) MgOAc 25mM Enzyme cofactor; sets rate of catalysis LiOAc 0.5 mM  Modifies rateof catalysis by competition with MgOAc KOAc 120 mM  Sets ionic strength;prevents protein aggregation DTT 40 mM Reducing agent; mitigates photo-induced damage MEA 10 mM Reducing agent; mitigates photo- induceddamage; TSQ cofactor DMAPA 2.5 mM  Anti-pause agent (increasespolymerase (pol) rate); TSQ cofactor Poly-dT 120 μM   Anti-stickingagent; increases accuracy & read length DMSO 4% Anti-sticking agent

Exemplary Formulation 3

TRIS buffer 30 mM Stabilizes reaction pH (pH 8.0) MgOAc 25 mM Enzymecofactor; sets rate of catalysis LiOAc 0.5 mM  Modifies rate ofcatalysis by competition with MgOAc KOAc 120 mM  Sets ionic strength;prevents protein aggregation MEA 10 mM Reducing agent; mitigates photo-induced damage; TSQ cofactor DMA 4% Anti-sticking agent

Exemplary Formulation 4

ACES buffer  67 mM Stabilizes reaction pH (pH 6.5) MnOAc  0.7 mM Enzymecofactor; sets rate of catalysis KOAc 120 mM Sets ionic strength;prevents protein aggregation DTT 120 mM Reducing agent; mitigates photo-induced damage DMF 4% Anti-sticking agent

As will be appreciated, the above formulations are not meant to belimiting, and one of skill in the art would readily understand that anysubset or combination of the above components is encompassed by thepresent invention. As with the general formulation, the above exemplaryformulations may further include any of the other additives discussedherein. In further embodiments, compositions of the invention includeone or more components of the above exemplary formulations incombination with other additives, including without limitation additivesfor photodamage mitigation, such as oxygen scrubbers and triplet statequenchers, and any of the additives described for example in U.S. Pat.No. 7,993,895; U.S. Patent Publication No. 20090325260; U.S. Pat. No.8,388,982; U.S. Patent Publication No. 20120052488; U.S. PatentPublication No. 20120009567 (01-012901); and U.S. ProvisionalApplication No. 61/707,621, filed Sep. 28, 2012, each of which is herebyincorporated by reference for all purposes and in particular for alldisclosure, figures or examples related to additives to include inreaction compositions.

Methods of the Invention

As will be appreciated, the present invention is applicable to a widevariety of analytical reactions where one wishes to monitor thatreaction by observing the specific association of a detectable reactioncomponent with another component, e.g., a surface bound component. Suchreactions include those where the association, itself, is the outcome ofinterest in the analysis, such as in binding assays, e.g.,antibody/antigen, hybridization assays, e.g., nucleic acid hybridizationassays, ligand/receptor binding assays, and incorporation assays, wherea labeled reactant is wholly or partly incorporated into a product ofthe reaction, thus rendering the product detectable.

Notably, however, the methods of the invention are also applicable toreactions where the association event is a transient prerequisite to thereaction of interest, but does not necessarily survive into the productof the reaction of interest. Such reactions include, for example, realtime reactions that observe the reactions of interest while they occur.Such reactions include, e.g., real time monitoring of polymerizationreactions, and the like, where the detectable component is not, itself,incorporated into the product. Such reactions include single moleculereal time detection of biological reactions such as nucleic acidreplication, translation and transcription, e.g., as described in Eid etal., Science Vol. 323 no. 5910 pp. 133-138 (January 2009), and Uemura etal., Nature 464, 987-988 (April 2010), each of which is herebyincorporated by reference in its entirety for all purposes and inparticular for all teachings related to single molecule real timedetection of biological reactions.

As will be appreciated, the observation of the association event ofinterest is rendered more difficult by the presence of interferingbackground signals presented by non-specifically associating detectablemolecules. In particular, if detectable reaction componentsnon-specifically interact with other components of the system so as tobe detectable, they may present signal events that are indistinguishablefrom relevant signal events, and would contribute to the overall signalprofile from which relevant signal events must be derived.

For example, the present invention is further illustrated with referenceto the monitoring of nucleic acid polymerization reactions that are usedin determining the nucleotide sequences of template or target nucleicacids. In one approach, a nucleic acid sequence is determined bydetecting the replication of that nucleic acid by a polymerase enzyme,which uses the underlying nucleic acid molecule as a template for thereplication process. In particular, a complex of a polymerase, atemplate nucleic acid, and a primer nucleic acid that is complementaryto a portion of the template, is provided immobilized upon a solidsupport, such as the surface of a transparent substrate. Nucleotidemonomers bearing a detectable label, such as an optically detectablefluorescent dye, are introduced to the complex, and if complementary tothe next nucleotide in the template, are incorporated by the polymerasein a primer extension reaction. By detecting when the labeled nucleotideis added, and identifying the type of nucleotide added, e.g., either byits spectrally distinct label or by virtue of having only introduced asingle type of nucleotide to the complex and continuing this processalong the template molecule, one can effectively read out the sequenceof the underlying template. These processes have been described using aserial and iterative process whereby only a single nucleotide or asingle type of nucleotide is added in the extension step, followed bywashing away excess unincorporated nucleotides and detection of wherethe label was incorporated. See, e.g., Metzker, Nature Reviews Genetics,11:31-46 (2010), which is hereby incorporated by reference in itsentirety for all purposes and in particular for all teachings related tosequencing.

Alternative processes monitor the extension reaction in a continuous,real-time approach, where the incorporation event is detected as itoccurs (rather than following a washing step). Such methods typicallyemploy processes that allow detection of the incorporation event even inthe presence of working concentrations of labeled nucleotides, bydetecting a characteristic signal that is associated with the actualincorporation event. For example, as described in Eid et al., supra, thecomplex is provided within an optically confined region thatilluminates, and thus observes, only fluorescently labeled compoundsthat are within a very small illumination region surrounding thecomplex. The result is that nucleotides that are going to beincorporated into the primer extension product are retained within theillumination volume for longer periods of time than randomly diffusing,unincorporated nucleotides. This extended presence provides a detectablesignal event that is attributable to a nucleotide incorporation event.Further, by providing the detectable label on a phosphate portion of thenucleotide, the label portion is liberated during the incorporationprocess, thus providing an end to the signal event when the labelportion diffuses out of the illumination volume.

In still other processes, the incorporation event is marked by thebringing together of interactive detectable groups, e.g., one on thenucleotide, and the other on the polymerase. When these groups are insufficient proximity as afforded by the incorporation process, theyyield a representative signal associated with the energy transferbetween the groups. See, e.g., U.S. Pat. No. 7,056,676, which is herebyincorporated by reference in its entirety for all purposes and inparticular for all teachings related to detection of nucleotideincorporation.

In each of the above processes, the nucleic acid polymerization complexis provided localized at or near a surface of a solid substrate. Suchlocalization may be through immobilization of the template or primernucleic acids to the substrate, or through immobilization of thepolymerase enzyme to the substrate. Further, in each process, detectionof an incorporation event is carried out by identifying a labelednucleotide either as it is reacting with the complex, or subsequent tothe reaction, when the labeled nucleotide is incorporated into theextended primer.

As will be appreciated, the presence of nonspecifically associatedfluorescent label groups, either as labeled nucleotide analogs that werenot incorporated, or as liberated fluorescently labeled by-products ofthe reaction, e.g., labeled polyphosphate groups, yields detectablesignal events that are not relevant to the reaction of interest, namelynucleotide incorporation. One of the major sources of these non-specificsignaling events is the adsorption of labeled groups to either the solidsubstrate or other immobilized components of the reaction, e.g., thepolymerase enzyme. Some processes have been employed to reduce thepotential for such non-specific adsorption to the underlying surfacethrough surface passivation processes (See, e.g., U.S. PatentApplication No. 2008-0176769, which is hereby incorporated by referencein its entirety for all purposes and in particular for all teachingsrelated to passivation processes). Even with such processes, somenon-specific binding can occur. For example, in some passivizedsurfaces, hydrophobic groups can remain on the underlying substratesurface that provides areas for non-specific adsorption of highlyhydrophobic organic fluorescent labeling groups. Likewise, presence ofextra charged groups may provide opportunities for association withoppositely charged detectable groups, in a non-specific fashion.Additives described herein can be used in the above-described processesto reduce and/or prevent non-specific adsorption, association or bindingof certain reactants, such as labeled nucleotide analogs, to otherreaction components.

The present invention is illustrated with reference to the followingnon-limiting examples.

EXAMPLES Example 1 Synthesis ofHeptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin

The synthetic scheme for Compound 1 is illustrated in FIG. 2. As per thescheme shown in FIG. 2, the target compound,Heptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin, (alsointerchangeably referred to as SBCD-MOR, Cyclo-Mor, andcyclodextrin-morpholino) (5), was synthesized in four steps startingfrom anhydrous β-cyclodextrin (1). Thus, commercially availableβ-cyclodextrin (1) was first dehydrated in a high vacuum oven at below100° C. to give the anhydrous material. The anhydrous 1 was then treatedwith TBDMSCI and imidazole in DMF to give the 6-hydroxy protectedsilylether 2 as shown in FIG. 2. Compound 2 was then reacted with sodiumhydride and N-(chloroacetyl)morpholine in DMF under nitrogen to give thedimorpholine substituted compound 3 in good yield. Deprotection of theTBDMS group in compound 3 was achieved with (n-Bu)4NF in THF.Sulfonation of compound 4 was carried out using sulfurtrioxide pyridinecomplex in DMF at room temperature. After neutralizing with 6 Npotassium hydroxide the crude product was dissolved in water and slowlyadded ethanol to it to induce the precipitation of potassium sulfatesalt. The inorganic salt was then filtered off and the desired product 5was obtained by crystallizing out from hot water and sufficient amountof ethanol.

Preparation of Anhydrous β-Cyclodextrin (1). The commercial hydratedβ-cyclodextrin was placed in Pyrex brand drying trays and subjected todehydration in an oven heated at 100° C. under high vacuum for at least48 hours until about 14% of weight of water was removed. The anhydrouspowder was then stored in a well sealed bottle.

Preparation of Heptakis(6-O-tert-butyldimethylsilyl)cyclomalto-heptaose(2). To an oven dried 2-L three-necked round bottom flask equipped witha football-shaped stir bar was added anhydrous N,N-dimethylformamide(DMF, 180 mL) under nitrogen atmosphere. The flask was heated usingheating mantle and solvent was brought to near boiling. Turned off theheat and removed the flask from the mantle. Added oven driedβ-cyclodextrin (89 g, 78.4 mmol) and stirred vigorously. Added imidazole(54.9 g, 806 mmol). Continued to stir vigorously until the solids werecompletely dissolved and the flask cooled to room temperature (total ofabout 30 min). A light honey-colored solution was obtained.

Slowly added dropwise a solution of tert-butyldimethylsilyl chloride(TBDMSCI, 86.86 g, 576.2 mmol) in anhydrous ethyl acetate (260 mL). Theaddition took approximately 3 h. The reaction was monitored with TLC(40:10:1 of CHCl3:MeOH:H2O). After stirring for 2 h additional TBDMSCI(9.09 g, 60.4 mmol) in EtOAc (26 mL) and imidazole (5.5 g) was addeddropwise. Stirred for 18 h at ambient temperature. [Note: AdditionalTBDMSCI and imidazole may be required to consume all the undersilylatedintermediates to the product. Small amount of oversilylatedside-products are generally presented in the reaction mixture.

Added EtOAc (1 L) and the resultant crystals (imidazole hydrochloride)were filtered off. Rinsed with EtOAc (2×100 mL). The combined filtratewas extracted and washed with acidified water (4×100 mL). After dryingover Na₂SO₄, the EtOAc layer was concentrated to small volume (˜400 mL).The crude product was collected through filtration, washed withEtOAc/hexanes (1:2, 2×100 mL) and dried in a desiccator under highvacuum to give 131.37 g of a white solid. Redissolved the solid in hotacetone (5 L) to give an oversaturated solution. Cooled slowly to bringupon the crystallization. Filtered to collect the solid (48.3 g). Secondcrop of solid (52.8 g) was obtained from the filtrate and a third cropof product (˜20 g) was obtained after reprocessing the second filtrate.On TLC, it revealed the first crop of product is pure and the second andthe third of products showed a major spot of product and a smallless-polar spot of oversilylated product.

Preparation ofHeptakis(6-O-tert-butyldimethylsilyl-2,3-diacetylmorpholine)-β-cyclodextrin(3). To an oven dried 500 mL three-neck round bottom flask connectedwith a condenser was charged 100 mL of tetrahydrofuran (THF) undernitrogen atmosphere. To it was slowly added sodium hydride (4.456 g, 186mmol) under nitrogen. After cease of bobbling the flask was placed in awater bath at 35° C., and a solution of 2 (11.0 g, 5.68 mmol) in THF (30mL) and N-(chloroacetyl)morpholine (35.36 g, 216.1 mmol) was addeddropwise through an addition funnel. The addition was complete in 10min. Stirring was continued for 2 h and reaction was monitored usingTLC. Added another 1 g of NaH to push the reaction to completion. Thereaction took a total of 14 days. Added iodomethane (5 mL) and stirredfor another 1 day. To the reaction mixture was slowly added dropwiseEtOH (8 mL) in 2 min to quench the reaction. Added n-butylacetate (100mL) and rotavapor evaporating off THF at reduced pressure untiln-butylacetate starting to distill over. Added EtOAc (100 mL) and thesuspension was placed in a centrifuged flask and centrifuged to separatethe solid from the solvent. Filtered to collect the solid using EtOAc,washed with EtOAc (2×50 mL) and dried. The crude product was used in thenext step without further purification.

Preparation of Heptakis(2,3-diacetylmorpholine)-β-cyclodextrin (4). To asolution of 3 in THF (75 mL) in a 250 mL round bottomed flask was addedt-Bu4NF (75 mL, 1 M in THF) and stirred for 70 h at room temperature.TLC (40:10:1 of CHCl₃:MeOH:water) showed the reaction was complete. Thesolvent was evaporated under reduced pressure, co-evaporated withacetonitrile (2×100 mL), dried, washed with hexanes (3×100 mL) anddecanted the solvent. After drying with high vacuum pump overnight therewas obtained the desired product 4. The crude product was used in thenext step without further purification.

Preparation of Heptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin(5). To a solution of 4 in DMF (150 mL) was added SO₃-pyridine complex(30.0 g, 188 mmol) and stirred for 18 hours at room temperature. LC/MSshowed formation of the product peak. The solution was then poured intoacetone (400 mL) and subjected to centrifugation to give the crudesolid. The supernatant was decanted and the gum was dissolved in water(100 mL). Added dropwise concentrated KOH until pH=10.5. Added 300 mL ofEtOH, the resultant inorganic salt was filtered off. The filtrate wasconcentrated to small volume (˜100 mL) and to it was added hot MeOH (500mL). After cooling to room temperature the resultant solid wascollected, dried in an oven under high vacuum at 45° C. overnight togive 16.3676 g of product 5 (yield for a total of three steps from 2:76.9%).

Example 2 Synthesis ofHeptakis(2,3-diacetyldiethylacetamide-6-sulfato)-β-cyclodextrin,(SBCD-DEA)

As shown in FIG. 3, the target compound,Heptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin, (SBCD-DEA)(8), was synthesized similarly in four steps starting from anhydrousβ-cyclodextrin (1) as per synthesis of SBCD-MOR. Compound 2 was thenreacted with sodium hydride and 2-chloro-N,N-diethylacetamide in DMFunder nitrogen to give the diacetyldiethylacetamide substituted compound6 in good yield. Deprotection of the TBDMS group in compound 6 wasachieved with (n-Bu)4NF in THF. Sulfonation of compound 7 was carriedout using sulfurtrioxide pyridine complex in DMF at room temperature.After neutralizing with 6 N potassium hydroxide the crude product wasdissolved in water and slowly added ethanol to it to induce theprecipitation of potassium sulfate salt. The inorganic salt was thenfiltered off and the desired product 8 was obtained by crystallizing outfrom hot water and sufficient amount of ethanol.

Preparation ofHeptakis(6-O-tert-butyldimethylsilyl-2,3-diacetyldiethyl-acetamide)-β-cyclodextrin(6). To an oven dried 500 mL three-neck round bottom flask connectedwith a condenser was charged 100 mL of tetrahydrofuran (THF) undernitrogen atmosphere. To it was slowly added sodium hydride (3.134 g,130.6 mmol) under nitrogen. After cease of bobbling the flask was placedin a water bath at 35° C., and a solution of 2 (7.00 g, 3.62 mmol) inTHF (20 mL) and 2-chloro-N,N-diethylacetamide (20 mL, 145.6 mmol) wasadded dropwise through an addition funnel. The addition was complete in10 min. Stirring was continued for 2 h and reaction was monitored usingTLC. Added another 1 g of NaH to push the reaction to completion. Thereaction took a total of 14 days. Added iodomethane (5 mL) and stirredfor another 1 day. To the reaction mixture was slowly added dropwiseEtOH (8 mL) in 2 min to quench the reaction. Added n-butylacetate (100mL) and rotavapor evaporating off THF at reduced pressure untiln-butylacetate starting to distill over. Added EtOAc (100 mL) and thesuspension was placed in a centrifuged flask and centrifuged to separatethe solid from the solvent. Filtered to collect the solid using EtOAc,washed with EtOAc (2×50 mL) and dried. The crude product was used in thenext step without further purification.

Preparation of Heptakis(2,3-diacetyldiethylacetamide)-β-cyclodextrin(7). To a solution of 6 in THF (60 mL) in a 250 mL round bottomed flaskwas added t-Bu4NF (75 mL, 1 M in THF) and stirred for 70 h at roomtemperature. TLC (40:10:1 of CHCl3:MeOH:water) showed the reaction wascomplete. The solvent was evaporated under reduced pressure,co-evaporated with acetonitrile (2×100 mL), dried, washed with hexanes(3×100 mL) and decanted the solvent. After drying with high vacuum pumpovernight there was obtained the desired product 6. The crude productwas used in the next step without further purification.

Preparation ofHeptakis(2,3-diacetyldiethylacetamide-6-sulfato)-β-cyclodextrin (8). Toa solution of 7 in DMF (120 mL) was added SO₃-pyridine complex (30.0 g,188 mmol) and stirred for 18 h at room temperature. LC/MS showedformation of the product peak. The solution was then poured into acetone(400 mL) and subjected to centrifugation to give the crude solid. Thesupernatant was decanted and the gum was dissolved in water (100 mL).Added dropwise concentrated KOH until pH=10.5. Added 300 mL of EtOH, theresultant inorganic salt was filtered off. The filtrate was concentratedto small volume (˜100 mL) and to it was added hot MeOH (500 mL). Aftercooling to room temperature the resultant solid was collected, dried inan oven under high vacuum at 45° C. overnight to give 10.1083 g ofproduct 8 (yield for a total of three steps from 2: 78.8%).

Example 3 Single Molecule, Real-Time DNA Sequencing with SulfatedCyclodextrins

Single molecule, real-time DNA sequencing was carried out using aPacific Biosciences® SMRT® Sequencing platform, see, e.g., Eid et al.,Science Vol. 323 no. 5910 pp. 133-138 (January 2009), where individualpolymerase/template/primer complexes are disposed within the observationvolume of zero mode waveguides in ZMW arrays. The reaction observes, inreal time, the reaction of the complexes with nucleoside polyphosphatesthat include detectable fluorescent labeling groups. Because thesegroups are labeled on the phosphate chain, the label groups are notincorporated into the primer extension product, and only the transientinteraction of the complex with the labeled portion of the nucleotide isdetected.

Four color, single molecule real time DNA sequencing reactions werecarried out using a standard SMRT sequencing protocols and reagents on aPacific Biosciences SMRT® Sequencing prototype system. Reactions werecarried out in the presence and absence ofheptakis(2,3-diacetylmorpholine-6-sulfato)-β-cyclodextrin at 120 μM.

FIG. 4 illustrates increased raw sequence read accuracy (percentage ofbases from the template that were accurately identified within a singlesequencing pass) from the test sequencing reaction that includes thecyclodextrin compound. As can be seen, the overall accuracy of thesystem is increased in the presence of the cyclodextrin compounds byapproximately 1 percentage point over the control. Without being boundto any particular theory of operation, it is believed that thenon-specific surface or other adsorption of labeled reagents to thesubstrate surface or the molecular complex increases error rate and thusdecreases the measured accuracy, which the cyclodextrin compounds serveto mitigate.

FIG. 5 shows a comparison of average read lengths obtained fromsequencing reactions in the presence and absence of the cyclodextrincompound. As can be seen, the test reaction demonstrates a markedincrease in mapped readlength (number of identified bases in acontiguous sequence from a single zero mode waveguide/reaction complex)for the template sequence used by 30%, or a total average readlengthincrease of approximately 170 bases. Accordingly, the addition of thecyclodextrin compounds provided marked improvements in at least twoimportant reaction quality metrics of readlength and accuracy.

Example 4 Screening Sulfonated Cholic Acids

In certain aspects, sulfonated cholic acids have similar effects tocyclodextrin compounds. Taurocholic acid (1×SO₃ ⁻) and a mixture of 2×,3×, and 4× sulfonated cholic acids were both tested.

Four-color sequencing with standard FCR chemistries was conducted underthe following conditions: 120 μM Cyclodextrin control, No additivenegative control, 120 μM SY427-31 (single SO₃ ⁻, 480 μM SY427-31, 120 μMSY427-29 (mixture), 480 μM SY427-29. (See FIG. 6 for an illustration ofthe compounds used).

Both cholic acid solutions acted similarly to cyclodextrin in theirability to reduce IPDs for FRET-labeled nucleotide analogs. Withoutbeing bound by theory, one possible mechanism of the effect of thesecompounds is by blocking analog binding sites. It appeared that SY427-29(mixture) required a higher concentration while SY427-31 (single SO₃ ⁻was effective at lower concentrations.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications, patents, patent applications, and/or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually and separately indicated to be incorporated by referencefor all purposes.

What is claimed is:
 1. A reaction mixture comprising: a first reactioncomponent coupled to a surface of a solid support, wherein the firstreaction component comprises a polymerase; a second reaction componenthaving a detectable property; a first additive that reduces interpulseduration (IPD), wherein said first additive comprises a member selectedfrom cholesterol, a cholic acid derivative, poly dT oligonucleotides,cyclodextrin, and a cyclodextrin derivative; a second additive thatreduces non-specific interactions between the first reaction componentand a surface upon which the reaction mixture is disposed, wherein thesecond additive comprises a member selected from an organic solvent,dimethylformamide (DMF), a detergent, and N-formylmorpholine (FMP); anda third additive that reduces polymerase pausing, wherein the thirdadditive comprises a member selected from a compound comprising anaromatic ring and two —NR₂ substituents, and an amino acid.
 2. Thereaction mixture of claim 1, wherein said cholic acid derivative istaurocholic acid.
 3. The reaction mixture of claim 1, wherein said aminoacid is arginine.
 4. The reaction mixture of claim 1, wherein saidsecond additive is DMF.
 5. The reaction mixture of claim 1, wherein saidsecond additive is FMP.
 6. The reaction mixture of claim 1, wherein saidcyclodextrin derivative is a sulfated cyclodextrin having the structure:

where n is from 6 to 12; R₁ is a group that is non-attractive to thesecond reaction component; R₂ and R₃ are associative groups to thesurface of the solid support; said sulfated cyclodextrin being presentat an isomeric purity of at least 80%; and detecting an interactionbetween the first reaction component and the second reaction componentby detecting the detectable property of the second reaction componentassociated with the first reaction component.
 7. The reaction mixture ofclaim 6, wherein R₁ is a sulfate group
 8. The reaction mixture of claim6, wherein R₂ and R₃ are selected from alkyl, acetamide, hydroxyalkyl,acyl, aryl or heteroaryl groups.
 9. The reaction mixture of claim 8,wherein R₂ and R₃ are selected from CH₂-acyl, diethylacetamide,dipropylacetamide, morpholino, piperazine, piperidine, pyrrolidine, andoxazolidine.
 10. The reaction mixture of claim 1, wherein the surfacecomprises a plurality of first reaction components, and wherein theplurality of first reaction components are provided disposed on thesurface as single molecules or single molecular complexes, each of whichis resolvable from others of the first reaction components disposed onthe surface.
 11. The reaction mixture of claim 10, wherein the pluralityof first reaction components are disposed within the observation volumeof zero mode waveguides.
 12. The reaction mixture of claim 1, whereinthe surface is a surface of a solid support, and further wherein thesolid support comprises a silica based substrate.
 13. The reactionmixture of claim 1, wherein the second reaction component comprises afluorescent labeling moiety.
 14. The reaction mixture of claim 1,wherein the second reaction component comprises a nucleotide analog. 15.The reaction mixture of claim 1, wherein the organic solvent is dimethylsulfoxide (DMSO) or dimethylacetamide (DMA).
 16. The reaction mixture ofclaim 1, wherein the compound comprising an aromatic ring and two —NR₂substituents is N,N-dimethyl-m-phenylenediamine (DMMP) orN,N-dimethyl-p-phenylenediamine (DMPP).
 17. The reaction mixture ofclaim 1, further comprising an additive for photodamage mitigation. 18.The reaction mixture of claim 17, wherein the additive for photodamagemitigation is selected from the group consisting of a triplet statequencher, an oxygen scrubber, dithiothreitol (DTT), andmercaptoethylamine (MEA).
 19. A system for conducting an analyticalreaction, the system comprising: a) a solid support comprising a firstsurface; b) a first reaction component coupled to the first surface ofthe solid support; and c) a reaction mixture disposed upon the firstsurface, the reaction mixture comprising a second reaction componenthaving a detectable property, a first additive that reduces interpulseduration (IPD), a second additive that reduces non-specific interactionsbetween the first reaction component and a surface upon which thereaction mixture is disposed, and a third additive that reducespolymerase pausing.
 20. The system of claim 19, wherein the firstreaction component comprises a polymerase; the first additive comprisesa member selected from cholesterol, a cholic acid derivative, poly dToligonucleotides, cyclodextrin, and a cyclodextrin derivative; thesecond additive comprises a member selected from an organic solvent,dimethylformamide (DMF), a detergent, and N-formylmorpholine (FMP); andthe third additive comprises a member selected from a compoundcomprising an aromatic ring and two —NR₂ substituents, and an aminoacid.